Emerging role of non-coding RNAs in the regulation of Sonic Hedgehog signaling pathway

Sonic Hedgehog (Shh) signaling cascade is one of the complex signaling pathways that control the accurately organized developmental processes in multicellular organisms. This pathway has fundamental roles in the tumor formation and induction of resistance to conventional therapies. Numerous non-coding RNAs (ncRNAs) have been found to interact with Shh pathway to induce several pathogenic processes, including malignant and non-malignant disorders. Many of the Shh-interacting ncRNAs are oncogenes whose expressions have been increased in diverse malignancies. A number of Shh-targeting miRNAs such as miR-26a, miR-1471, miR-129-5p, miR-361-3p, miR-26b-5p and miR-361-3p have been found to be down-regulated in tumor tissues. In addition to malignant conditions, Shh-interacting ncRNAs can affect tissue regeneration and development of neurodegenerative disorders. XIST, LOC101930370, lncRNA-Hh, circBCBM1, SNHG6, LINC‐PINT, TUG1 and LINC01426 are among long non-coding RNAs/circular RNAs that interact with Shh pathway. Moreover, miR-424, miR-26a, miR-1471, miR-125a, miR-210, miR-130a-5p, miR-199b, miR-155, let-7, miR-30c, miR-326, miR-26b-5p, miR-9, miR-132, miR-146a and miR-425-5p are among Shh-interacting miRNAs. The current review summarizes the interactions between ncRNAs and Shh in these contexts.


Introduction
Sonic Hedgehog (Shh) signaling cascade is one of the complicated signaling pathways that administrate the accurately controlled developmental processes in multicellular organisms. It has an important role in the establishment of the outlines of cellular differentiation to regulate multifaceted organ formation. This pathway affects these cellular processes via a cascade that changes the equilibrium between activator and repressor types of glioma-associated oncogene (Gli) transcription factors.
A number of Hedgehog (Hh) ligands as well as Patched receptors, Smoothened receptor, Suppressor of fused homolog, Kif7, PKA and cAMP participate in the transfer of signals to the Gli transcription factors. Transfer of the activator form of Gli to the nucleus and its binding with the promoters of target genes lead to the stimulation of the transcription of these genes [1]. Hh gene has been firstly discovered about four decades ago via genetic screen experiments in Drosophila [2].
Shh participates in the tissue regeneration processes and repair mechanism in the post-embryonic period. This pathway has a crucial role in the induction of diverse populations of neurons in the central nervous system, governing several morphogenetic processes in this system [3].
Abnormal regulation of these signals has been shown to be associated with congenital malformations, aberrant tissue regeneration, stem cell renewal and carcinogenesis [4]. Expression, cellular uptake and translocation of the Shh protein as a key Hh ligand precursor have important effects in the regulatory function of Shh signaling [5]. Two other Hedgehog homologues, namely Desert (Dhh) and Indian (Ihh) have been identified in mammals.
Functional studies have shown the importance of Shh signaling in ventral cell type induction. Moreover, disruption of this pathway and recessive mutations have led to cyclopia and severe holoprosencephaly in mice, respectively [6]. In human, heterozygote mutations in Shh have been associated with different clinical features of holoprosencephaly [7].
Shh signaling is also implicated in the regulation of function of normal adult stem cells as well as cancer stem cells [8]. Dysregulation of the Hh signaling pathway has been linked with developmental abnormalities including Gorlin syndrome [9] and cancer [10,11]. Abnormal activity of this pathway is also involved in the tumor formation and induction of resistance to radio/chemotherapy [12]. Thus, efforts have been made to find novel Shh signaling inhibitors to combat these features [12].
More recently, numerous non-coding RNAs (ncR-NAs) have been found to interact with Shh pathway to induce several pathogenic processes, including malignant and non-malignant disorders. The current review summarizes the interactions between ncRNAs and Shh in these contexts. Long non-coding RNAs (lncRNAs), microRNAs (miRNAs) and circular RNAs (circRNAs) are three main regulatory ncRNAs which are discussed in this context. LncRNAs are transcripts with sizes more than 200 nt that regulate expression of genes at different levels. They can regulate chromatin function, influence the assemblage and functions of membraneless nuclear bodies, control the stability and expression of cytoplasmic mRNAs and interfere with signaling pathways [13]. miRNAs have about 22 nt and mainly affect gene expression at post transcriptional level [14]. Finally, circRNAs are made by either typical spliceosome-mediated or lariat-type splicing. They can regulate expression of genes through different mechanisms [15].
For the purpose of preparation of the current review, we searched Google Scholar and PubMed databases with the key words "Shh signaling" OR "Sonic Hedgehog" AND "lncRNA" OR "miRNA" OR "circRNA". Then, we assessed the abstract of retrieved articles to validate their relevance with the topic. We included studies that assessed function of ncRNAs in cell lines, animal model or clinical samples. A total of 50 studies were included in this review article.

Non-malignant disorders
The importance of interactions between ncRNAs and Shh pathway has been assessed in different cell lines. This type of interaction has been found to be implicated in the pathophysiology of alopecia. This speculation is based on the results of three-dimensional culture of dermal papilla cells, a group of cells that induce regeneration of hair follicles. Experiments in this type of culture have verified up-regulation of XIST lncRNA and Shh and down-regulation of miR-424. Mechanistically, XIST has been found to sponge miR-424 to increase Shh expression. XIST silencing has led to inhibition of activity of dermal papilla cells, suppression of their proliferation and reduction of ALP activity. In fact, XIST silencing has inhibited Shh mediated hedgehog signals through affecting expression of miR-424 [16].

Human development
Shh-interacting ncRNAs are also involved in the developmental processes. For instance, serum response factor (SRF) controls lineage specification of embryonic stem cell progenitor cells through miR-210-mediated gene silencing. Up-regulation of miR-210 in murine embryonic stem cells-originated embryoid bodies has suppressed cell growth and blocked expression of cardiac progenitor proteins Nkx2.5 and Gata4 and terminal differentiated contractile markers Mlc2v and βMHC. On the other hand, miR-210 silencing has led to activation of cardiac progenitor gene. The effect of miR-210 is exerted through decreasing activity of Shh signaling, which nurtures the cardiac progenitor program. Mechanistically, miR-210 silences Shh activity through targeting 3' UTR of Shh transcript [17]. Activation of Shh/Gli1 signaling pathway through miR-130a-5p/Foxa2 axis has been shown to affect development of fetal lung, thus being involved in the pathogeensis of congenital diaphragmatic hernia [18]. miR-199 is another miRNA that participate in craniofacial development through modulation of Shh pathway [19].

Cancer
The interaction between ncRNAs and Shh signaling pathway has also been assessed in cancer cell lines. For instance, the tumor suppressor miRNA miR-26a has been found to be down-regulated in breast cancer cell lines. Up-regulation of miR-26a has led to blockade of cell proliferation, clone formation capacity and metastatic aptitude of breast cancer cells, and induction of sensitivity to docetaxel. miR-26a could directly target FAM98A. Up-regulation of this miRNA has resulted in down-regulation of FAM98A, SHH, SMO and GLI1. Taken together, miR-26a suppresses breast carcinogenesis through inhibiting expression of FAM98A, and decreasing activity of Shh pathway [20]. miR-1471 is another down-regulated miRNA in breast cancer cells. This miRNA has been found to be sponged by LOC101930370. LOC101930370 silencing has suppressed progression of breast cancer, while inhibition of miR-1471 has increased aggressive and metastatic abilities of MCF-7 cells. Furthermore, expression levels of SHH and Gli-1 have been significantly decreased following LOC101930370 silencing, and increased by miR-1471 inhibition. Cumulatively, LOC101930370 has been found to increase expression of SHH through sponging miR-1471 [21]. Another study has revealed dysregulation of several lncRNAs in Twist-positive mammosphere cells in breast cancer cell lines. Notably, the Shh-GLI1-related lncRNA-Hh has been among these lncRNAs. Expression of this lncRNA is regulated by Twist. Moreover, lncRNA-Hh can directly target GAS1 to induce Hh activity, which in turn enhances expression of GLI1, and increases SOX2 and OCT4 levels to regulate maintenance of cancer stem cells. The latter is reflected in enhancement of mammosphere-formation efficiency and self-renewal ability in cell lines. Knock down of lncRNA-Hh in Twist-positive breast cancer cells has attenuated activity of Shh-GLI1 signaling and decreased levels of SOX and OCT4 [22]. CircBCBM1 is another example of ncRNAs that can promote metatstatic ability of breast cancer cells through acting as a molecular sponge for miR-125a and modulating expression of BRD4. This circRNA also upregulate MMP9 levels through enhancing activity of Shh pathway [23]. Figure 1 shows the role of Shh-interacting ncRNAs in breast cancer.
In pancreatic cancer cell lines, miR-132 expression has been found to be up-regulated parallel with downregulation of Shh levels. Besides, miR-132 mimics could significantly decrease expression of Shh at both transcript and protein levels, facilitating proliferation of pancreatic cancer cells, which has been accompanied by down-regulation of Cyclin-D1, cleaved Caspase-3/9, and suppression of cell apoptosis [24].
Shh-interacting ncRNAs can also affect the pathogenesis of brain tumors. For instance, miR-326 can effectively suppress proliferation, and induce apoptosis in glioma cells via attenuating the activation of the SHH/GLI1 pathway [25]. Moreover, miR-9 via targeting PTCH1 and enhancing expression of GLI1 can trigger the activation of Shh cascade and affect expression of drug efflux transporters, MDR1 and ABCG2 in glioblastoma cells, therefore enhancing Temozolomide resistance in tumor cells [26]. Figure 2 shows the role of Shh-interacting miRNA in glioma/glioblastoma. Additional in vitro studies have revealed participation of Shh-related ncRNAs in several developmental processes as well as carcinogenic processes (Table 1).

Animal studies
Animal studies have shown participation of Shh-related ncRNAs in a variety of non-neoplastic disorders, namely acute myocardial infarction, alopecia, cerebrovascular disorders, diabetes mellitus, inflammatory bowel disease, lung fibrosis, osteoporosis, Parkinson's disease and trigeminal neuralgia as well as different types of cancers ( Table 2). For instance, knock-down of miR-802-5p has resulted in reduction of cell apoptosis after myocardial infarction through enhancing activity of Shh signaling, thus decreasing myocardial injury and improving cardiac function [57]. Moreover, experiments in animal models have shown up-regulation of XIST increases dermal papilla cells-mediated hair follicle regeneration via targeting miR-424 to promote Shh expression, thus activating hedgehog signaling [16]. Moreover, miR-153 has been found to decrease expression of PTC expression and enhance activity of Shh signaling pathway to increase angiogenesis in a rat model of cerebral ischemic injury [58].
Experiments in animal models of breast cancer have verified that knock-down of lncRNA-Hh [22] and circB-CBM1 [23] can led to reduction of tumor growth. Meanwhile, miR-326 has been shown to increase effects of curcumin in animal models of glioblastoma through modulation of Shh/GLI1 signaling pathway [25].

Studies in clinical samples
Expression assays in clinical samples from a variety of tumor types have indicated down-regulation of Shh-targeting miRNAs such as miR-26a [20] and miR-1471 [21] in breast cancer, miR-129-5p in cervical cancer [28], miR-361-3p in retinoblastoma, miR-26b-5p in gallbladder carcinoma [33], and miR-361-3p in retinoblastoma [53]. In gallbladder cancer, dysregulation of miR-26b-5p has been associated with age and sex of patients, tumor invasion, differentiation degree, tumor location, and TNM stage [33]. Conversely, an expression assay in pancreatic cancer samples has shown up-regulation of miR-132 and downregulation of Shh [24]. In neuroblastoma samples, downregulation of CDON and up-regulation of miR-181-a and miR-181-b have been associated with poor overall survival, higher tumor stage and more aggressive phenotype [64].

Discussion
Shh signaling is involved in a variety of cellular functions, including tissue development and regeneration, stem cell functions and carcinogenesis. Thus, it is not surprising that ncRNAs that regulate activity of this pathway are implicated in the pathogenesis of a wide range of human disorders. In fact, this pathway represents a Mounting researches suggest that dysregulation of sonic hedgehog signaling pathway can play a key role in tumorigenesis in breast cancer cells. As an illustration, the recent study has detected that overexpression of lncRNA-Hh can activate Shh-GLI1 signaling and promote the expression levels of SOX2 and OCT4, thereby enhancing cancer stem cells generation in Twist-positive breast cancer cells [22]. Further experiment has validated that lncRNA LOC101930370 can significantly elevate SHH and Gli-1 expression via sponging miR-1471, therefore promoting cell proliferation and metastasis in breast cancer cells by modulating the hedgehog cascade [21]. Moreover, another research has pointed out that miR-26a has a remarkable part in suppressing breast cancer cell proliferation and invasion via downregulating the expression levels of FAM98A, SHH, SMO and GlI1, thereby inactivating the sonic hedgehog pathway in tumor cells [20] prototype of shared pathways between embryogenesis and carcinogenesis.
In the context of malignant disorders, Shh-interacting ncRNAs not only affect cancer progression, but also determine response of cancer cells to a variety of anticancer therapies. Both functions can be explained by the crucial roles of this pathway in the induction of stemness. However, at least in some types of cancers, Shh signaling seems to have protective effects against carcinogenesis. For instance, in pancreatic cancer, expression of the Shhtargeting miRNA miR-132 has been found to be up-regulated parallel with down-regulation of Shh levels [24]. Accumulating evidence has revealed that upregulation of miR-326 in combination with curcumin can effectively contribute in the suppression of proliferation, and elevation of the apoptosis process in glioma cells via attenuating the activation of the SHH/GLI1 pathway [25]. Another finding confirms that miR-9 via targeting PTCH1 and promoting expression of GLI1 can trigger the activation of sonic hedgehog cascade and modulate expression of drug efflux transporters, MDR1 and ABCG2 in glioblastoma cells, therefore enhancing Temozolomide resistance in tumor cells [26]. Furthermore, mounting research has demonstrated that Chidamide can play an important role in inhibiting the expression levels of Shh, Ihh, and Dhh via upregulating miR-338-5p, thereby suppressing the growth rate, migration, and invasion of human malignant glioma cells. In fact, Chidamide exerts its effects by enhancing oxidative stress via the miR-338-5p-mediated regulation of Hedgehog pathway [27]  MGPC Müller glia-derived progenitor cells, DCs dendritic cells, OA Osteoarthritis, EMT epithelial-mesenchymal transition, ∆ knock-down, deletion, TCRV triacetyl resveratrol, TMZ temozolomide, MFE mammosphereformation efficiency, LUAD lung adenocarcinoma Table 2 Interaction between ncRNAs and Shh pathway based on studies in animal models
It is estimated that one-third of malignancies are correlated with abnormal activity of the Hh signaling pathway [70]. Dysregulation of Hh signaling can contribute to the imitation, growth, metastasis, and apoptosis of several types of cancers. In fact, three patterns of induction of the Hh signaling cascade have been identified in several cancers. These patterns are ligand independent oncogenic Hh pathway, autocrine or juxtacrine and paracrine or reverse paracrine patterns [70].
Moreover, there are several examples of interactions between two classes of ncRNAs in the context of regulation of activity of Shh signals. XIST/miR-424, LOC101930370/miR-1471, circBCBM1/miR-125a, TUG1/miR-132, SNHG6/miR-26b-5p, LINC-PINT/ miR-425-5p, and SNHG6/miR-135a-5p are examples of lncRNA/miRNA or circRNA/miRNA pairs that cooperatively regulate activity of Shh pathway. The regulatory impact of these axes on function of Shh pathway should be assessed in different cellular and disease contexts to find whether they act in a context-specific manner or a ubiquitous manner. This has importance in design of novel therapies for each disorder in which abnormal function of Shh pathway has been detected. However, circRNAs have limited roles as miRNA sponges in most cases [71][72][73][74]. In fact, most circRNAs are much less abundant than miRNAs and are not predicted to function as miRNA sponges [75,76].
Although Shh-related ncRNAs are expected to influence the prognosis and clinical outcome of cancer, this issue has been verified only in the gallbladder cancer, neuroblastoma and laryngeal cancer. Thus, future studies should assess the prognostic roles of these ncRNAs as well as their potential as diagnostic markers.
A number of anti-cancer agents such as propofol have been found to exert their effects through modulation of Shh-related ncRNAs. This agent has been found to simultaneously decrease activity of Shh and PI3K/AKT pathways [48]. Therefore, modulation of expression of Shh-related ncRNAs is a promising anticancer strategy.
Taken together, the data presented above indicates contribution of several ncRNAs in the regulation of Shh pathway and their involvement in the pathogenesis of several disorders. However, this data has some limitations. No comprehensive assessments of different types of ncRNAs using next generation sequencing techniques have been performed. Thus, the interactive networks between different types of ncRNAs and Shh signaling components have not been identified yet.
A number of Hh inhibitors, namely Smo antagonist, Cyclopamine, Sulforaphane, Baicalein, Sangunarine, GANT61, Sonidegib, and PF-04449913 have been used for inhibition of cancer stem cells [77]. Meanwhile, activity of cancer stem cells has been shown to be affected by a number of mentioned ncRNAs. Therefore, combination of mentioned therapeutic modalities with ncRNA-targeted therapies might be regarded as effective options for eradication of cancer stem cells.
Since Shh-related ncRNAs have fundamental roles in the pathogenesis of human disorders, it is possible to down-regulate or up-regulate their expression in order to alter the pathological events in the course of disease evolution. In order to translate the basic science about the role of ncRNAs in the regulation of Shh pathway into clinical application, the following steps should be followed: (1) comprehensive assessment of expression of different classes of ncRNAs in clinical samples; (2) application of system biology methods for analysis of the acquired data; (3) understanding the complex network between different classes of ncRNAs and components of Shh pathway; (4) establishment of in vitro and